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MethMotif (https://methmotif.org) is a publicly available database that provides a comprehensive repository of transcription factor (TF)-binding profiles, enriched with DNA methylation patterns. In this release, we have enhanced the platform, expanding our initial collection to over 700 position weight matrices (PWM), all of which include DNA methylation profiles. One of the key advancements in this release is the segregation of TF-binding motifs based on their cofactors and DNA methylation status. We have previously demonstrated that gene ontology (GO) enriched terms associated with TF target genes may differ based on their association with alternative cofactors and DNA methylation status. MethMotif provides precomputed GO annotations for each human TF of interest, as well as for TF-co-TF complexes, enabling a comprehensive analysis of TF functions in the context of their co-factors. Additionally, MethMotif has been updated to encompass data for two new species, Mus musculus and Arabidopsis thaliana, widening its applicability to a broader community. MethMotif stands out as the first and only TF-binding motifs database to incorporate context-specific PWM coupled with epigenetic information, thereby enlightening context-specific TF functions. This enhancement allows the community to explore and gain deeper insights into the regulatory mechanisms governing transcriptional processes.
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Metilación de ADN , Bases de Datos Genéticas , Factores de Transcripción , Animales , Humanos , Ratones , Sitios de Unión , Anotación de Secuencia Molecular , Motivos de Nucleótidos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
A recent paper by Jiang et al. in BMC Bioinformatics presented guidelines on long-read sequencing settings for structural variation (SV) calling, and benchmarked the performance of various SV calling tools, including NanoVar. In their simulation-based benchmarking, NanoVar was shown to perform poorly compared to other tools, mostly due to low SV recall rates. To investigate the causes for NanoVar's poor performance, we regenerated the simulation datasets (3× to 20×) as specified by Jiang et al. and performed benchmarking for NanoVar and Sniffles. Our results did not reflect the findings described by Jiang et al. In our analysis, NanoVar displayed more than three times the F1 scores and recall rates as reported in Jiang et al. across all sequencing coverages, indicating a previous underestimation of its performance. We also observed that NanoVar outperformed Sniffles in calling SVs with genotype concordance by more than 0.13 in F1 scores, which is contrary to the trend reported by Jiang et al. Besides, we identified multiple detrimental errors encountered during the analysis which were not addressed by Jiang et al. We hope that this commentary clarifies NanoVar's validity as a long-read SV caller and provides assurance to its users and the scientific community.
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Benchmarking , Simulación por Computador , GenotipoRESUMEN
Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP. Once the DNA is labeled, it can be isolated for NGS library preparation. We termed this method as universal NicE-seq (nicking enzyme-assisted sequencing). We also demonstrate a single tube method that enables direct NGS library preparation from low cell numbers without DNA purification. Furthermore, we demonstrated universal NicE-seq on FFPE tissue section sample.
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Cromatina , ADN , ADN/genética , Nucleosomas , Mapeo Cromosómico/métodos , Análisis de Secuencia de ADN/métodos , Formaldehído , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
The Ets transcription factor PU.1 is essential for inducing the differentiation of monocytes, macrophages, and B cells in fetal liver and adult bone marrow. PU.1 controls hematopoietic differentiation through physical interactions with other transcription factors, such as C/EBPα and the AP-1 family member c-Jun. We found that PU.1 recruits c-Jun to promoters without the AP-1 binding sites. To address the functional importance of this interaction, we generated PU.1 point mutants that do not bind c-Jun while maintaining normal DNA binding affinity. These mutants lost the ability to transactivate a target reporter that requires a physical PU.1-c-Jun interaction, and did not induce monocyte/macrophage differentiation of PU.1-deficient cells. Knock-in mice carrying these point mutations displayed an almost complete block in hematopoiesis and perinatal lethality. While the PU.1 mutants were expressed in hematopoietic stem and early progenitor cells, myeloid differentiation was severely blocked, leading to an almost complete loss of mature hematopoietic cells. Differentiation into mature macrophages could be restored by expressing PU.1 mutant fused to c-Jun, demonstrating that a physical PU.1-c-Jun interaction is crucial for the transactivation of PU.1 target genes required for myeloid commitment and normal PU.1 function in vivo during macrophage differentiation.
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Hematopoyesis , Factor de Transcripción AP-1 , Animales , Sitios de Unión , Diferenciación Celular/genética , Hematopoyesis/genética , Ratones , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun , Factor de Transcripción AP-1/genéticaRESUMEN
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second leading cause of cancer worldwide. Despite approvals of several therapeutics to treat advanced HCC in the past few years, the impact of anti-angiogenic treatment on HCC patient overall survival remains limited. This suggests there may be alternative, perfusion-independent roles of endothelial cells that support tumor progression. Thus, we leveraged a well-defined hydrogel system to establish co-culture models to mimic and characterize the angiocrine crosstalk between HCC and endothelial cells in vitro. Co-cultures of HCC cell lines or patient-derived xenograft organoids with endothelial cells exhibited the upregulation of MCP-1, IL-8 and CXCL16, suggesting that the HCC-endothelial interactions established in our models recapitulate known angiocrine signaling. Additionally, by subjecting co-cultures and mono-cultures to RNA sequencing, transcriptomic analysis revealed an upregulation in the expression of genes associated with tumor necrosis factor (TNF) signaling, such as that of chemokines, suggesting that endothelial cells induce HCC cells to generate an inflammatory microenvironment by recruiting immune cells. Finally, HCC-endothelial angiocrine crosstalk in the co-culture models polarized macrophages towards a pro-inflammatory and pro-angiogenic phenotype, paralleling a tumor-associated macrophage subset previously reported in HCC. Together, these findings suggest that these HCC-endothelial co-culture models may serve as important models to understand and target the interplay between angiogenesis and the immune milieu.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Humanos , Neoplasias Hepáticas/patología , Organoides/metabolismo , Microambiente TumoralRESUMEN
Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.
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Transportadoras de Casetes de Unión a ATP , Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Humanos , Regiones no Traducidas 3' , Transportadoras de Casetes de Unión a ATP/genética , Sordera/genética , Pérdida Auditiva/genética , Pérdida Auditiva Sensorineural/genética , Mutación , Linaje , Fosfolípidos/metabolismo , Sitios de Empalme de ARNRESUMEN
The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.
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Neoplasias Encefálicas , Factor de Transcripción E2F1 , Glioblastoma , Glioma , Histonas , Factor de Transcripción STAT3 , Acetilación , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/metabolismo , Histonas/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Glioblastoma (GBM) is a uniformly lethal disease driven by glioma stem cells (GSCs). Here, we use a chemical biology approach to unveil previously unknown GBM dependencies. By studying sulconazole (SN) with anti-GSC properties, we find that SN disrupts biotin distribution to the carboxylases and histones. Transcriptomic and metabolomic analyses of SN-treated GSCs reveal metabolic alterations that are characteristic of biotin-deficient cells, including intracellular cholesterol depletion, impairment of oxidative phosphorylation, and energetic crisis. Furthermore, SN treatment reduces histone biotinylation, histone acetylation, and expression of superenhancer-associated GSC critical genes, which are also observed when biotin distribution is genetically disrupted by holocarboxylase synthetase (HLCS) depletion. HLCS silencing impaired GSC tumorigenicity in an orthotopic xenograft brain tumor model. In GBM, high HLCS expression robustly indicates a poor prognosis. Thus, the dependency of GBM on biotin distribution suggests that the rational cotargeting of biotin-dependent metabolism and epigenetic pathways may be explored for GSC eradication.
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CCCTC binding factor (CTCF) is an important factor in the maintenance of chromatin-chromatin interactions, yet the mechanism regulating its binding to chromatin is unknown. We demonstrate that zinc finger protein 143 (ZNF143) is a key regulator for CTCF-bound promoter-enhancer loops. In the murine genome, a large percentage of CTCF and ZNF143 DNA binding motifs are distributed 37 bp apart in the convergent orientation. Furthermore, deletion of ZNF143 leads to loss of CTCF binding on promoter and enhancer regions associated with gene expression changes. CTCF-bound promoter-enhancer loops are also disrupted after excision of ZNF143. ZNF143-CTCF-bound promoter-enhancer loops regulate gene expression patterns essential for maintenance of murine hematopoietic stem and progenitor cell integrity. Our data suggest a common feature of gene regulation is that ZNF143 is a critical factor for CTCF-bound promoter-enhancer loops.
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Factor de Unión a CCCTC/metabolismo , Elementos de Facilitación Genéticos , Células Madre Hematopoyéticas/metabolismo , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Animales , ADN/metabolismo , Hematopoyesis/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Unión Proteica , Estabilidad Proteica , Transcripción GenéticaRESUMEN
The lack of cancer-associated fibroblasts (CAFs) in patient-derived organoid (PDO) models is a major limitation as CAFs contribute to tumor progression and drug resistance. In the present study, we addressed this problem by establishing in vitro conditions that enable the co-culture of colorectal cancer (CRC) PDO with patient-derived CAFs. Considering that the CRC extracellular matrix is high in hyaluronan and collagen I, we hypothesized that hyaluronan-gelatin hydrogels may serve as a suitable alternative 3D matrix to traditionally used basement membrane extracts to support the co-culture of CRC PDO and CAFs. We report the development of in vitro models consisting of CRC PDO encapsulated within a well-defined three-dimensional (3D) hyaluronan-gelatin hydrogel and co-cultured with patient-derived CAFs. Through RNA- and whole -exome sequencing, we first show that these hydrogels are capable of maintaining key molecular characteristics of the original patient tumors in CRC PDO but not support the culture of CAFs. Further, based on our findings that CRC PDO culture medium poorly supports CAF viability, we developed a co-culture strategy that maintains the viability of both CRC PDO and CAFs. We found that even in the absence of growth factors conventionally used to support CRC PDO culture, CAFs were able to maintain the proliferation of the cultured CRC PDO in the hydrogels and restore distinct biological pathways absent in the PDO culture alone but present in patient tissues. Lastly, we demonstrate that these CRC PDO-CAFs co-culture models are suitable for evaluating standard-of-care drugs, making them potentially very useful for realizing personalized cancer medicine. STATEMENT OF SIGNIFICANCE: We report the development of an engineered tumor microenvironment consisting of colorectal cancer patient-derived organoids (CRC PDO) encapsulated within a well-defined three-dimensional (3D) hyaluronan-gelatin hydrogel and co-cultured with patient-derived cancer-associated fibroblasts (CAFs). Through sequential culture, we found that in the absence of growth factors added to the co-culture, CAFs were able to maintain the proliferation of the cultured CRC PDO in the hydrogels and restore distinct biological pathways absent in the PDO culture alone but present in patient tissues. Lastly, we demonstrate that these CRC PDO-CAFs models are suitable for evaluating standard-of-care drugs, making them potentially very useful for realizing personalized cancer medicine.
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Neoplasias Colorrectales , Organoides , Proliferación Celular , Técnicas de Cocultivo , Humanos , Hidrogeles , Microambiente TumoralRESUMEN
Tumor-specific metabolic rewiring, acquired to confer a proliferative and survival advantage over nontransformed cells, represents a renewed focus in cancer therapy development. Hepatocellular carcinoma (HCC), a malignancy that has hitherto been resistant to compounds targeting oncogenic signaling pathways, represents a candidate cancer to investigate the efficacy of selectively antagonizing such adaptive metabolic reprogramming. To this end, we sought to characterize metabolic changes in HCC necessary for tumorigenesis. We analyzed gene expression profiles in three independent large-scale patient cohorts who had HCC. We identified a commonly deregulated purine metabolic signature in tumors with the extent of purine biosynthetic enzyme up-regulation correlated with tumor grade and a predictor of clinical outcome. The functional significance of enhanced purine metabolism as a hallmark in human HCC was then validated using a combination of HCC cell lines, patient-derived xenograft (PDX) organoids, and mouse models. Targeted ablation of purine biosynthesis by knockdown of the rate-limiting enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) or using the drug mycophenolate mofetil (MMF) reduced HCC proliferation in vitro and decreased the tumor burden in vivo. In comparing the sensitivities of PDX tumor organoids to MMF therapy, we found that HCC tumors defined by high levels of IMPDH and guanosine nucleosides were most susceptible to treatment. Mechanistically, a phosphoinositide 3-kinase (PI3K)-E2F transcription factor 1 (E2F1) axis coordinated purine biosynthetic enzyme expression, deregulation of which altered the activity of mitogen-activated protein kinase/RAS signaling. Simultaneously abolishing PI3K signaling and IMPDH activity with clinically approved inhibitors resulted in greatest efficacy in reducing tumor growth in a PDX mouse model. Conclusion: Enhanced purine metabolic activity regulated by PI3K pathway-dependent activation of E2F1 promotes HCC carcinogenesis, suggesting the potential for targeting purine metabolic reprogramming as a precision therapeutic strategy for patients with HCC.
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The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.
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Granulocitos/metabolismo , Mielopoyesis , Receptores del Factor Estimulante de Colonias/biosíntesis , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Regulación hacia Arriba , beta Catenina/metabolismo , Animales , Candida albicans , Candidiasis/genética , Candidiasis/metabolismo , Ratones , Ratones Transgénicos , Receptores del Factor Estimulante de Colonias/genética , Factores de Transcripción TCF/genética , beta Catenina/genéticaRESUMEN
Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.
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Autorrenovación de las Células/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Lisina Acetiltransferasa 5/genética , Transactivadores/genética , Animales , Biomarcadores , Ciclo Celular , Diferenciación Celular/genética , Daño del ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histonas/metabolismo , Lisina Acetiltransferasa 5/metabolismo , Ratones , Transporte de Proteínas , Transactivadores/metabolismoRESUMEN
RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress-induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Hematopoyéticas/fisiología , Leucemia/genética , Metiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Animales , Apoptosis/genética , Supervivencia Celular/genética , Familia , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patología , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Mutación Missense , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , LinajeRESUMEN
The recent advent of third-generation sequencing technologies brings promise for better characterization of genomic structural variants by virtue of having longer reads. However, long-read applications are still constrained by their high sequencing error rates and low sequencing throughput. Here, we present NanoVar, an optimized structural variant caller utilizing low-depth (8X) whole-genome sequencing data generated by Oxford Nanopore Technologies. NanoVar exhibits higher structural variant calling accuracy when benchmarked against current tools using low-depth simulated datasets. In patient samples, we successfully validate structural variants characterized by NanoVar and uncover normal alternative sequences or alleles which are present in healthy individuals.
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Pruebas Genéticas/métodos , Variación Estructural del Genoma , Leucemia Mieloide/genética , Secuenciación de Nanoporos/métodos , Análisis de Secuencia de ADN/métodos , Células Cultivadas , Pruebas Genéticas/normas , Células HCT116 , Humanos , Leucemia Mieloide/patología , Secuenciación de Nanoporos/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/normasRESUMEN
Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.
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Quinasa 5 Dependiente de la Ciclina/genética , Citidina/análogos & derivados , Dioxigenasas/genética , Neurogénesis/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Diferenciación Celular/genética , Citidina/genética , Citidina/metabolismo , Metilación de ADN/genética , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/genética , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Factores del Dominio POU/genética , Fosforilación , Regiones Promotoras GenéticasRESUMEN
Transcription factors (TFs) are sequence-specific DNA binding proteins, fine-tuning spatiotemporal gene expression. Since genomic occupancy of a TF is highly dynamic, it is crucial to study TF binding sites (TFBSs) in a cell-specific context. To date, thousands of ChIP-seq datasets have portrayed the genomic binding landscapes of numerous TFs in different cell types. Although these datasets can be browsed via several platforms, tools that can operate on that data flow are still lacking. Here, we introduce TFregulomeR (https://github.com/benoukraflab/TFregulomeR), an R-library linked to an up-to-date compendium of cistrome and methylome datasets, implemented with functionalities that facilitate integrative analyses. In particular, TFregulomeR enables the characterization of TF binding partners and cell-specific TFBSs, along with the study of TF's functions in the context of different partnerships and DNA methylation levels. We demonstrated that TFs' target gene ontologies can differ notably depending on their partners and, by re-analyzing well characterized TFs, we brought to light that numerous leucine zipper TFBSs derived from ChIP-seq experiments documented in current databases were inadequately characterized, due to the fact that their position weight matrices were assembled using a mixture of homodimer and heterodimer binding sites. Altogether, analyses of context-specific transcription regulation with TFregulomeR foster our understanding of regulatory network-dependent TF functions.
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Biología Computacional , Proteínas de Unión al ADN/genética , ADN/genética , Factores de Transcripción/genética , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica/genética , Genoma/genética , Unión Proteica/genéticaRESUMEN
Intron retention (IR) by alternative splicing is a conserved regulatory mechanism that can affect gene expression and protein function during adult development and age-onset diseases. However, it remains unclear whether IR undergoes spatial or temporal changes during different stages of aging or neurodegeneration like Alzheimer's disease (AD). By profiling the transcriptome of Drosophila head cells at different ages, we observed a significant increase in IR events for many genes during aging. Differential IR affects distinct biological functions at different ages and occurs at several AD-associated genes in older adults. The increased nucleosome occupancy at the differentially retained introns in young animals suggests that it may regulate the level of IR during aging. Notably, an increase in the number of IR events was also observed in healthy older mouse and human brain tissues, as well as in the cerebellum and frontal cortex from independent AD cohorts. Genes with differential IR shared many common features, including shorter intron length, no perturbation in their mRNA level, and enrichment for biological functions that are associated with mRNA processing and proteostasis. The differentially retained introns identified in AD frontal cortex have higher GC content, with many of their mRNA transcripts showing an altered level of protein expression compared to control samples. Taken together, our results suggest that an increased IR is an conserved signature that is associated with aging. By affecting pathways involved in mRNA and protein homeostasis, changes of IR pattern during aging may regulate the transition from healthy to pathological state in late-onset sporadic AD.
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Envejecimiento/genética , Enfermedad de Alzheimer/genética , Intrones/genética , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Drosophila , Humanos , Ratones , TranscriptomaRESUMEN
Since publication of this article, the authors identified an error in the acknowledgments section. The original sentence in acknowledgments reads as follows.
RESUMEN
Several recent studies have portrayed DNA methylation as a new player in the recruitment of transcription factors (TF) within chromatin, highlighting a need to connect TF binding sites (TFBS) with their respective DNA methylation profiles. However, current TFBS databases are restricted to DNA binding motif sequences. Here, we present MethMotif, a two-dimensional TFBS database that records TFBS position weight matrices along with cell type specific CpG methylation information computed from a combination of ChIP-seq and whole genome bisulfite sequencing datasets. Integrating TFBS motifs with TFBS DNA methylation better portrays the features of DNA loci recognised by TFs. In particular, we found that DNA methylation patterns within TFBS can be cell specific (e.g. MAFF). Furthermore, for a given TF, different DNA methylation profiles are associated with different DNA binding motifs (e.g. REST). To date, MethMotif database records over 500 TFBSs computed from over 2000 ChIP-seq datasets in 11 different cell types. MethMotif portal is accessible through an open source web interface (https://bioinfo-csi.nus.edu.sg/methmotif) that allows users to intuitively explore the entire dataset and perform both single, and batch queries.